Protease activation has been implicated as an early signaling event associated with interleukin-1B (IL-l) Inhibition of pancreatic B-cell function. IL-1 Inhibits insulin secretion from pancreatic B-cells by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide.IL-1 also induces the co-expression of the inducible isoform of cyclooxygenase (COX-2) that results in the over production of proinflammatory prostaglandins. The current studies were designed to study the involvement of protease(s) in the signaling pathway of IL-1-induced NOS and COX-2 expression using a serine protease inhibitor, Na-p-tosy1-L-lysline chloromethyl ketone (TLCK) and a proteasome inhibitor, MG I32. TLCK inhibits IL-1-induced nitrite formation (an oxidation product of nitric oxide) by dispersed rat islet cells and RINm5F insulinoma cells in a concentration-dependent manner, with complete inhibition observed at 100 uM. TLCK (100 uM) also inhibits iNOS gene expression at the level of mRNA and protein synthesis, suggesting that the site of action of TLCK is prior to transcription. Our recent studies suggest that the transcription factor, NFkB, is essential for activation of iNOS gene expression TLCK (100 uM) blocks IL-I-Induced translocation of NFkB from cytosol to the nucleus in RINm5F cells based on electrophoretic mobility shift assays. The activation of NFkB is proposed to involve activation of the ubiquitin-proteasome complex. We show that a proteasome inhibitor, MG I32, inhibits IL-1-induced nitric oxide production in a concentration-dependent manner with an 1C50 of - 1 uM. Similar to TLCK, MG 132 inhibits iNOS gene expression at the level of mRNA and protein synthesis. MG 132 also inhibits cytokin-induced nitrite and PG2 production by human islets. We further show that IL-I-induced COX-2 expression and prostaglandlin production are completely blocked by 100 uM TLCK or 10 uM MG 132. These results indicate that IL-1-induced iNOS and Cox-2 expression by human and rat islets share a common signaling pathway in utilizing proteasome and NFkB and suggest strategies of intervention that may prevent the over production of their inflammatory products.